Rod-Derived cone viability factor (RdCVF) is a newly discovered survival factor that prolongs the health and life of cone photoreceptors. This factor has attracted considerable interest because it could potentially maintain health of cells in patients with a diverse set of retinal degenerative diseases, including retinitis pigmentosa (RP) and age-related macular degeneration (AMD). It is likely that this factor will exert maximal therapeutic effect if it is produced constantly by cells neighboring the cone photoreceptors, such as retinal pigment epithelial (RPE) cells. One approach to achieve this effect is to deliver the cDNA encoding RdCVF to these cells via a recombinant viral vector such as adeno-associated virus (AAV). This proposal aims to provide the foundation for testing the ability of AAV-mediated delivery of RdCVF to delay retinal degeneration in a diverse set of animal models. In Aim 1, I will use AAV vectors targeting RPE and photoreceptor cells to deliver RdCVF in vitro and verify appropriate transgene expression. I will then test its ability to promote cone viability, using a novel organ culture system in which primary RPE cells infected with AAV.RdCVF are co-cultured with degenerating all-cone retinal explants. In Aim 2, I will inject these vectors subretinally into transgenic mice with cone and rod degeneration, and look for photoreceptor rescue and funtional preservation. I will also perform studies comparing localization and expression levels of RdCVF in mouse versus human retinas, and will examine rod-degenerative human retinas for diminution of RdCVF expression as is seen in mice. These experiments will establish a stable RdCVF delivery system in vivo and assess its viability effects in a variety of forms of retinal degeneration, and will provide information about expression patterns critical for translation of these studies into human applications. In Aim 3, I will examine the role of the hydrophobic patch exposed on the short form of RdCVF in mediating its cone viability effects; I will also explore its role in mediating RdCVF multimerization, an effect hypothesized based on preliminary data. I will perform these analyses using deletion and site-directed mutagenesis, and hope to glean vital mechanistic information about the protective effects of RdCVF on photoreceptors. In sum, this project will provide important data about the biological activity of the cone-protective neurotrophic factor RdCVF and the efficacy of virally-mediated constitutive retinal expression of this protein on degenerating photoreceptors. This work could ultimately pave the way for a human clinical trial testing the ability of AAV-mediated delivery of RdCVF to prevent blindness in inherited or acquired forms of retinal degeneration. [unreadable] [unreadable] [unreadable]